[Construction of plant expressive vector of human beta-defensin-2 gene].

OBJECTIVE

Several evidences suggested that transgenic plants would be a facile and economic bioreactor for large-scale production of industrial and pharmaceutical recombinant proteins. This study is made in an attempt to establish plant bioreactor for expression of recombinant hBD-2.

METHODS

Recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was inserted into a plant expressive vector-pCAMBIA1304, which closely located the down-stream of CaMV35S promoter. Agrobacterium tumefaciens LBA4404 was transformed with the recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His. The positive clones of LBA4404 transformed by rpCAMBIA1304/hBD-2/His were selected on a culture plate containing kanamycin. The callus tissues were transfected by positive clones of LBA4404, and positive callus were examined by using the resistant selection of hygromycin gene.

RESULTS

The evidences of enzyme digestion, PCR and sequence analysis confirmed that recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was correctly inserted into pCAMBIA1304 and was located between CaMV35S promoter and Nos terminal cordon to construct recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His, thus indicating that rpCAMBIA1304/hBD-2/His has successfully transformed Agrobacterium tumefaciens LBA4404 and positive clones have been isolated. The results from resistant selection of hygromycin gene showed that rpCAMBIA1304/hBD-2/His has been transferred into the callus of wheat, and the differentiation of callus tissue under selective pressure of hygromycin is carried out continually.

CONCLUSION

The above data suggest that the technique of transgenic plant is workable for the production of recombinant hBD-2.